Gene hp dna transfection reagent

Manufactured by Roche

Sourced in Germany

GENE HP DNA Transfection Reagent is a laboratory product designed for the delivery of DNA into mammalian cells. It facilitates the introduction of genetic material into the cell for various research applications.

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Lab products found in correlation

Acat1, by Merck Group (2 mentions) Hpa004428, by Merck Group (2 mentions) Sc 376841, by Santa Cruz Biotechnology (2 mentions) β catenin, by Santa Cruz Biotechnology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) E cadherin, by Cell Signaling Technology (2 mentions) Vimentin, by Cell Signaling Technology (2 mentions) 5 aza dc, by Merck Group (2 mentions) Pcmv6 entry vector, by OriGene (2 mentions) Flow cytometry, by BD (1 mentions) Plasmid mini kit, by Qiagen (1 mentions) Steponeplus system, by Thermo Fisher Scientific (1 mentions) First stand reverse transcription system, by Transgene (1 mentions)

5 protocols using gene hp dna transfection reagent

Overexpression of ACAT1 in NPC cells

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Antibody to proteins of interest, source and dilutions used were as follows: ACAT1 (1:1000, HPA004428, Sigma, USA), β-catenin (1:1000, sc-376841, Santa Cruz, USA) and E-cadherin (1:1000, #3195P), Vimentin (1:1000, #5741P) and GAPDH (1:10000 #5174P) were purchased from Cell Signaling Technology.
The demethylating reagent 5-aza-dC was purchased from Sigma (#A3656, USA). Full-length ACAT1 cDNA (Origene, USA) was subcloned into the pCMV6-Entry vector (Origene, USA). 2μg pCMV6-Entry or pCMV6-ACAT1 plasmids were used in a 48 hrs transfection protocol using an overnight culture of NPC cells at 70–90% confluence in 6-well dishes, using an X-treme GENE HP DNA Transfection Reagent (Roche, Germany).

Lu Y., Zhou X., Zhao W., Liao Z., Li B., Han P., Yang Y., Zhong X., Mo Y., Li P., Huang G., Xiao X., Zhang Z, & Zhou X. (2021). Epigenetic Inactivation of Acetyl-CoA Acetyltransferase 1 Promotes the Proliferation and Metastasis in Nasopharyngeal Carcinoma by Blocking Ketogenesis. Frontiers in Oncology, 11, 667673.

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Lentiviral Delivery of miR-375 in Colon Cancer

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Lentiviral particles were produced by co-transfection of HEK293 cells. Cells were cultured at the density of ∼70% in 10 cm tissue culture dishes. Recombinant pCDH-puro-miR-375 or pCDH-puro empty vector with packaging plasmid PMD2. G were added into each dish to set up teatment and control group respectively, helping with x-treme GENE HP DNA Transfection Reagent (Roche, CH). The virus particles were collected 48 h after transfection, and then infected with HT29, HCT116 and CaCo2 cells respectively, supplemented with 12 μg.mL^-1 of infection reagent polybrene. Infected cells expressing green fluorescence were detected by flow cytometry (BD, US) to evaluate the infection efficiency, and selected miR-375 stable expressing cells using puromycin.

Wei R., Yang Q., Han B., Li Y., Yao K., Yang X., Chen Z., Yang S., Zhou J., Li M., Yu H., Yu M, & Cui Q. (2017). microRNA-375 inhibits colorectal cancer cells proliferation by downregulating JAK2/STAT3 and MAP3K8/ERK signaling pathways. Oncotarget, 8(10), 16633-16641.

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Transient Transfection of TMEM16A in HEK293T and LA795 Cells

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HEK293T and LA795 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Sijiqing), 100 IU/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator at 37 °C with 5% CO₂. The cells were transiently transfected with cDNA for mouse TMEM16A (Accession Number NM_178642.5) using x-treme GENE HP DNA Transfection Reagent (Roche). Cells were seeded on 12-mm glass coverslips in a 24-multiwell plate. Following transfection, the cells were maintained in Dulbecco's modified Eagle's medium at 37 °C for 24 h before patch recording. The shRNA-16A plasmid was transfected into cells with the above transfection reagent to knockdown the expression of endogenous TMEM16A in LA795 cells. The following shRNA targeting the mouse TMEM16A gene was used: CCTGCTAAACAACATCATT (2399–2418 nt).

Shi S., Ma B., Sun F., Qu C, & An H. (2021). Theaflavin binds to a druggable pocket of TMEM16A channel and inhibits lung adenocarcinoma cell viability. The Journal of Biological Chemistry, 297(3), 101016.

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Plasmid Transfection Protocol for Cell Culture

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Before transfection, the highly pure plasmids were prepared using Plasmid Mini Kits (Qiagen, Germany). Cells in log phase were cultured in 6-well plates or 24-well plates for approximately 24 h before transfection. Each well was transfected with plasmids and X-treme GENE HP DNA Transfection Reagent (Roche) mixture, which was mixed in 200 μl antibiotic-free and serum-free medium according to manufacturer’s instruction. Transfection medium was removed and medium containing antibiotic and serum were added after 6 h. The mixture was then cultured for 72 h.

Liu T.H., Dong X.L., Pan C.X., Du G.Y., Wu Y.F., Yang J.G., Chen P., Lu C, & Pan M.H. (2016). A newly discovered member of the Atlastin family, BmAtlastin-n, has an antiviral effect against BmNPV in Bombyx mori. Scientific Reports, 6, 28946.

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Investigating ACAT1 in NPC Cells

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The information of antibodies was as followed: ACAT1 (1:1000, HPA004428, Sigma, USA), β-catenin (1:1000, sc-376841, Santa Cruz, USA) and E-cadherin (1:1000, #3195P), Vimentin (1:1000, #5741P) and GAPDH (1:10000 #5174P) were purchased from Cell Signaling Technology.
The demethylating reagent 5-aza-dC was purchased from Sigma (#A3656, USA). Full-length cDNA from the open reading frame of ACAT1 (Origene, USA) was subcloned into the pCMV6-Entry vector (Origene, USA). NPC cells were cultured in 6-well dishes to 70-90% con uence then transfected with 2μg pCMV6-Entry or ACAT1 plasmid using an X-treme GENE HP DNA Transfection Reagent (Roche, Germany) for 48 hrs.
Real-time RT-PCR Brie y, rst-strand complementary DNA was synthesized using a First-Stand Reverse Transcription System (Transgene, Beijing, China). Real-time RT-PCR was carried out using SYBR Green PCR master mix in Step One Plus System (Applied Biosystems, USA). The primer sequences and cycling conditions for all experiments were as follows, ACAT1-F 5'-GGCTGGTGCAGGAAATAAGA-3', ACAT1-R 5' GGAATCCCTGCCTTTTCAAT-3'; GAPDH-F 5'-GCTCAGACACCATG-GGGAAG-3', GAPDH-R 5'-TGTAGTTGAGGTCAATGAAGGGG-3'. Empty vector-transfected cells were used as control. The relative gene expression was calculated using the comparative threshold cycle (2 -△△CT ) equation. All the experiments were performed in triplicate.

Lu Y., Zhou X., Zhao W., Liao Z., Li B., Yang Y., Zhong X., Mo Y., Li P., Huang G., Xiao X., Zhang Z., & Zhou X. (2020). Epigenetic Inactivation of Acetyl-Co A acetyltransferase 1 promotes the proliferation and metastasis of nasopharyngeal carcinoma via decreasing ketogenesis.

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